Biology Module 2

• Microscopy


  The use of microscopes to analyse cell components and observe organelles
• Magnification


  How many times bigger the image produced by the microscope is than the real-life object you are viewing
• Resolution


  The ability to distinguish between objects that are close together (i.e. the ability to see two structures that are very close together as two separate structures)
• Types of microscopes


  • Optical (light) microscopes
  • Electron microscopes
  • Laser scanning confocal microscopes
• Optical (light) microscopes


  • Use light to form an image
  • Have a maximum resolution of around 0.2 micrometres (µm) or 200 nm
  • Can be used to observe eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts
  • Cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes
  • Have a maximum useful magnification of about ×1500
• Electron microscopes


  • Use electrons to form an image
  • Have a maximum resolution of around 0.0002 µm or 0.2 nm (around 1000 times greater than that of optical microscopes)
  • Can be used to observe small organelles such as ribosomes, the endoplasmic reticulum or lysosomes
  • Have a maximum useful magnification of about ×1,500,000
• Types of electron microscopes

  • Transmission electron microscopes (TEMs)
  • Scanning electron microscopes (SEMs)
• Transmission electron microscopes (TEMs)


  • Use electromagnets to focus a beam of electrons
  • The beam of electrons is transmitted through the specimen
  • Denser parts of the specimen absorb more electrons, making these denser parts appear darker on the final image produced
  • Give high-resolution images (more detail)
  • Can only be used with very thin specimens or thin sections of the object being observed
  • Cannot be used to observe live specimens
  • Do not produce a colour image
• Scanning electron microscopes (SEMs)

  • Scan a beam of electrons across the specimen
  • The beam bounces off the surface of the specimen and the electrons are detected, forming an image
  • Can be used on thick or 3-D specimens
  • Allow the external, 3-D structure of specimens to be observed
  • Give lower resolution images (less detail) than TEMs
  • Cannot be used to observe live specimens
  • Do not produce a colour image
• Laser scanning confocal microscopes

  • The cells being viewed must be stained with fluorescent dyes
  • A thick section of tissue or small living organisms are scanned with a laser beam
  • The laser beam is reflected by the fluorescent dyes
  • Multiple depths of the tissue section/organisms are scanned to produce an image
  • Can be used on thick or 3-D specimens
  • Allow the external, 3-D structure of specimens to be observed
  • Produce very clear images due to the laser beam being focused at a very specific depth
  • It is a slow process and takes a long time to obtain an image
  • The laser has the potential to cause photodamage to the cells
• Optical microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied
• Key components of an optical microscope

  • Eyepiece lens
  • Objective lenses
  • Stage
  • Light source
  • Coarse and fine focus
• Other tools used with optical microscopes

  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette
  • Staining solution
• Preparing a slide using a liquid specimen


  1. Add a few drops of the sample to the slide using a pipette

  2. Cover the liquid/smear with a coverslip and gently press down to remove air bubbles

  3. Wear gloves to ensure there is no cross-contamination of foreign cells
• Preparing a microscope slide using a solid specimen

  1. Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

  2. Use scissors to cut a small sample of the tissue

  3. Peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using a scalpel or forceps)

  4. Apply a stain

  5. Gently place a coverslip on top and press down to remove any air bubbles
• Preparing a microscope slide using a solid specimen (alternative method)

  1. Treat the tissue sample with chemicals to kill/make the tissue rigid

  2. Fix the specimen using formaldehyde (preservative), dehydrate it using a series of ethanol solutions, impregnate it in paraffin/resin for support then cut thin slices from the specimen using a microtome

  3. Remove the paraffin from the slices/specimen, apply a stain and mount using a resin

  4. Gently place a coverslip on top and press down to remove any air bubbles
• Preparing a microscope slide using a solid specimen (alternative method)

  1. Freeze the specimen in carbon dioxide or liquid nitrogen

  2. Cut the specimen into thin slices using a cryostat

  3. Place the specimen on the slide and add a stain

  4. Gently place a coverslip on top and press down to remove any air bubbles
• When using an optical microscope always start with the low power objective lens
• Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration
• Switching to the lower power objective lens and using the coarse focus can help get a clearer image if the image is unclear or blurry
• Graticule


  A small disc that has an engraved ruler, which can be placed into the eyepiece of a microscope to act as a ruler in the field of view
• Using a graticule to take measurements of cells


  1. The graticule must be calibrated for the objective lens that is in use, using a stage micrometer

  2. Once calibrated, the graticule can be used as a ruler in the field of view
• The size of cells or structures of tissues may appear inconsistent in different specimen slides due to the 3D nature of cells and tissues being cut at different planes
• Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that cannot be seen
• The treatment of specimens when preparing slides could alter the structure of cells
• Staining in light microscopy

  • Coloured dyes are used to make naturally transparent tissues visible under the microscope
  • The dyes absorb specific colours of light while reflecting others, making the stained structures visible
• Common stains used in light microscopy

  • Toluidine blue
  • Phloroglucinol
• Staining for electron microscopy

  Heavy-metal compounds are used as dyes to cause the tissues to show up black or different shades of grey, as electrons have no colour
• Stains used for electron microscopy

  • Osmium tetroxide
  • Ruthenium tetroxide
• Any colour present in electron micrographs is not natural and is added using image-processing software

  
• Cell surface membrane


  Controls the exchange of materials between the internal cell environment and the external environment
• Cell surface membrane


  • Formed from a phospholipid bilayer
  • Diameter of around 10 nm
• Cell wall


  • Freely permeable to most substances
  • Provides structural support to the cell
• Cell wall


  • Found in plant cells but not in animal cells
  • Formed of the polysaccharide cellulose in plants, and peptidoglycan in most bacterial cells
• Plasmodesmata


  Narrow threads of cytoplasm (surrounded by a cell membrane) that connect the cytoplasm of neighbouring plant cells
• Nucleus


  Contains chromatin (a complex of DNA and histone proteins) which is the genetic material of the cell
• Nucleus


  • Present in all eukaryotic cells (except red blood cells)
  • Separated from the cytoplasm by a double membrane (the nuclear envelope) which has many pores
  • Contains chromatin (the material from which chromosomes are made)
  • Contains one or more darkly stained regions called nucleoli which are the sites of ribosome production
• Mitochondria


  The site of aerobic respiration within all eukaryotic cells
• Mitochondria


  • Surrounded by double-membrane with the inner membrane folded to form cristae
  • The matrix formed by the cristae contains enzymes needed for aerobic respiration, producing ATP
  • Contain small circular pieces of DNA (mitochondrial DNA) and ribosomes
• Chloroplasts


  Found in the green parts of a plant, the green colour is a result of the photosynthetic pigment chlorophyll